FTM Lecture 19 - Use of Genomics in Diagnosis.pdf

Basic Principles of Medicine 1 Module: Foundations to Medicine Lecture 19 Lecture Title: Use of Genomics in Diagnosis Name of Lecturer: Dr. Mary Maj, PhD St Georges University © Copyright All year 1 courses materials, whether in print or online, are protected by...

Basic Principles of Medicine 1 Module: Foundations to Medicine Lecture 19 Lecture Title: Use of Genomics in Diagnosis Name of Lecturer: Dr. Mary Maj, PhD St Georges University © Copyright All year 1 courses materials, whether in print or online, are protected by copyright. The work, or parts of it, may not be copied, distributed or published in any form, printed, electronic or otherwise. As an exception, students enrolled in year 1 of St. George’s University School of Medicine and their faculty are permitted to make electronic or print copies of all downloadable files for personal and classroom use only, provided that no alterations to the documents are made and that the copyright statement is maintained in all copies. View only files, such as lecture recordings, are explicitly excluded from download and creating copies of these recordings by students and other users are strictly illegal. The author of this document has made the best effort to observe current copyright law and the copyright policy of St George's University. Users of this document identifying potential violations of these regulations are asked to bring their concern to the attention of the author. Pre-Lecture - Required Reading and Objectives 1. You can find the following pages and chapters on your Sakia site under: Book Access FTM Genomics in Diagnosis Readings from Korf textbook which includes: Methods 1.1 – Isolation of DNA – p4 Methods 2.2 – Polymerase chain reaction – p35 Methods 4.1 – DNA Sequencing – p68 Methods 4.3 – Gene Chips – p83 Methods 4.4 – Next Generation Sequencing p84 Hot Topic 4.1 – Genome Sequencing p 85 (note that this also defines the exome) Methods 6.1 – Chromosomal Analysis – p105 Methods 6.3 – FISH – p108 Methods 6.4 – Genomic Microarrays – P109 Clinical Snapshot 6.3 – 22q11.2 Deletion Syndrome – p120 Hot Topic 6.1 – Pathology Associated with CNV – p122 Clinical Snapshot 4.1 Huntington Disease – pg74 2. DLA 17 now one recording and one set of notes SOM.MK.III.BPM1.1.FTM.3.GNET.0069 Explain how triplet repeat expansion disorders are diagnosed SOM.MK.III.BPM1.1.FTM.3.GNET.0070 Discuss why each triplet repeat expansion disorder needs its own unique test for diagnosis SOM.MK.III.BPM1.1.FTM.3.GNET.0071 Explain how a G-band karyotype is prepared SOM.MK.III.BPM1.1.FTM.3.GNET.0072 Describe FISH and how it can be used in diagnostic applications SOM.MK.III.BPM1.1.FTM.3.GNET.0073 Compare and contrast : Metaphase FISH, Interphase FISH, and Chromosome painting (SKY FISH, or spectral karyotype) SOM.MK.III.BPM1.1.FTM.3.GNET.0074 Describe the strategy behind the microarray: CGH Microarray, SNP Microarray, cDNA Microarray  Objectives for Lecture & DLA: Genomics in diagnosis SOM.MKIII.BPM1.1.FTM.3.GNET.0069 Explain how triplet repeat expansion disorders are diagnosed SOM.MKIII.BPM1.1.FTM.3.GNET.0070 Discuss why each triplet repeat expansion disorder needs its own unique test for diagnosis SOM.MKIII.BPM1.1.FTM.3.GNET.0071 Explain how a G-band karyotype is prepared SOM.MKIII.BPM1.1.FTM.3.GNET.0072 Describe FISH and how it can be used in diagnostic applications Compare and contrast : Metaphase FISH, Interphase FISH, and Chromosome painting (SKY FISH, or spectral SOM.MKIII.BPM1.1.FTM.3.GNET.0073 karyotype) SOM.MKIII.BPM1.1.FTM.3.GNET.0074 Describe the strategy behind the microarray: CGH Microarray, SNP Microarray, cDNA Microarray SOM.MKIII.BPM1.1.FTM.3.GNET.0075 Analyze genomic approaches that are used in the diagnosis of human disorders SOM.MKIII.BPM1.1.FTM.3.GNET.0076 Analyze how PCR can be used to obtain a diagnosis of a genetic disorder SOM.MKIII.BPM1.1.FTM.3.GNET.0077 Discuss how next generation sequencing is being used in genomic applications Describe the difference between whole genome sequencing (WGS), whole exome sequencing (WES), gene SOM.MKIII.BPM1.1.FTM.3.GNET.0078 panels, and single targeted gene sequencing Describe the different types of mutations that may be observed in the human genome and explain how they are SOM.MKIII.BPM1.1.FTM.3.GNET.0079 categorized. Consider the relative pros and cons of the genomic and genetic techniques described in this section, and their SOM.MKIII.BPM1.1.FTM.3.GNET.0080 limits of resolution Understanding the principles and applications of genetic testing techniques is essential for medical professionals. It enables accurate interpretation of test results and informed decision-making of patient care 1  Detection of Triplet Repeat Expansion Disorders · threshold Huntington disease (HD)- autosomal dominant Myotonic dystrophy type 1 (MD1)-autosomal dominant Hitrected Fragile X syndrome (FX)- X linked Friedrich ataxia (FA)- autosomal recessive 5’ BE * CGG FX- FMR1 AUG GAA FA- FXN CAG HD- HTT TAA CTG MD- DMPK 3’ - Only detected by PCR amplification of the area and sizing: 1. Electrophoresis after PCR affected technique 2. Column chromatography after PCR Each disorder must have its own unique primers specific for gene and region of mutation & 2 SOM.MKIII.BPM1.1.FTM.3.GNET.0069 & 70 Other uses of PCR in Diagnosis Forensic analysis Viral, bacterial and fungal infections If targeted to a specific region, DNA can be amplified for sequencing, RFLP analysis, forensic analysis, prenatal diagnosis 3 SOM.MKIII.BPM1.1.FTM.3.GNET.0076 Direct Methods 1: Hypothesis driven Make a probe to hybridize your target Southern blot, Southern-RFLP- ssDNA probe 300-1000 bp - ASO blot- ssDNA probe 15-30 bp - PCR-RFLP, PCR sizing- ssDNA probes are a pair of primers 15-30 bp FISH- ssDNA probes 10,000 - to 20,000 bp Interphase FISH Northern- ssRNA or ssDNA about 100 bp Western- Antibody Sanger sequencing- ssDNA primer 15-30 bp Metaphase FISH 5 SOM.MKIII.BPM1.1.FTM.3.GNET.0075  Direct Methods 2: Query Entire Genome look atcelsmetaphase > N - Karyotype: G-banding Microarray: Comparative Genome Hybridization (CGH), Single Nucleotide Polymorphism (SNP), Expression Array ~ turn RNA into - > - Spectral Karyotype (SKY, paint each chromosome a different color) DNA D Next Generation Sequencing: obtaining sequence in multiplex fashion Can't see deletion Whole Genome Sequencing (WGS) Exomes of known genes (whole exome sequencing, WES) A panel of related genes (e.g., all known genes involved in cardiomyopathy) One gene with in-depth read (sequence one gene about 50 times) 6 SOM.MKIII.BPM1.1.FTM.3.GNET.0075  Link this concept: Allelic vs. Locus Heterogeneity Allelic Heterogeneity Locus Heterogeneity Same gene eg : CTFR gene Mutated Different genes Different mutations on same gene Different genes have mutations leading to same syndrome Example: Cystic Fibrosis: AR inheritance Example: Treacher Collins: AR or AD inheritance CFTR gene: Common mutations shown Mutations leading to non-functional proteins C 3120+1G->A p. del F508 TCOF1 on chromosome 5 POLR1C on chromosome 6 POLR1D on chromosome 13 CFTR Mutation F508 Severe phenotype CFTR Mutation p.G970D Moderate-severe phenotype TCOF1 Treacher Collins – AD CFTR Mutation c.3120 +1G>A Moderate-severe phenotype POLR1D Treacher Collins – AD CFTR Mutation p.R117H Mild phenotype POLR1C Treacher Collins – AR ……..many others 8 SOM.MKIII.BPM1.1.FTM.3.GNET.0045 Genomic Tests- All about the resolution microscopic ~ 3Mb Ch. 15 Next Generation Sequencing G-banding FISH Array CGH Euchromatic Genome Deletions, limits depend one base pair at a time Whole on size of probe Euchromatic Genome chromosome G-banding deletions or 10-1000 Kb to detect “Sequencing by synthesis” aneuploidy deletions Resolution depends on duplications if >0.5 million base pairs how dense the chip is, Resolution depends on Trisomy banding pattern is (0.5Mb) 244K and 400K = What you are querying 21 affected Microdeletion disorders: 244,000 probes or by reference library Count: 47 >5 million base DiGeorge (137 kb) 400,000 probes Whole Genome (WGS) chromosomes Prader-Willi (125 kb) pairs Angelman (180 kb) Whole Exome (WES) Would give space between Panel of candidate genes Williams (215 kb) the probes as 8.9 kb and 5.3 (5Mb) Wolf-Hirschhorn (98 kb) One gene: in-depth read kb respectively (50-100x) Cri du chat (561 kb) 9 Some cancers SOM.MKIII.BPM1.1.FTM.3.GNET.0075 Karyotype Analysis Developed in 1950’s Prepared from lymphocyte cell culture arrested in metaphase, fixed to a slide and stained with Geimsa stain (AT rich regions) Chromosomes from one cell called Chromosome or Metaphase Spread Can see chromosome number, large translocations, large insertions and deletions (gains or losses) Rule of thumb: deletions or duplications >5Mb are usually seen Deletions or duplications too small to be seen are 10 Mb - deletion, not seen by karyotype ↑ deletion Chr. 11 >7 Mb g - duplication , not seen by karyotype insertion dication 17 SOM.MKIII.BPM1.1.FTM.3.GNET.0074 lay gain Microarray has probes of reference DNA attached to a glass chip, Each spot has many of the same DNA is probe attached sheared and a fluorescent probe attached Hybridization +1 0 -1 Copy Number Variant Patient Loss Equal hybridization of More Control DNA More Patient DNA patient DNA & control hybridizes than Patient hybridizes than Control 19 DNA = No CNV = patient LOSS = patient GAIN Emery's Elements of Medical Genetics and Genomics, 5, 51-66 SOM.MKIII.BPM1.1.FTM.3.GNET.0074  SNP Microarray Design Probe to hybridize patient DNA fixed on glass slide (3 different SNP loci Selected Polymorphisms by array manufacturer: rs100000010 rs1108985 rs1109052 shown) To have A allele or B allele Will show 3 clusters for majority of people AA & AB & BB Probe=strands of DNA fixed on a slide (hundred thousand to millions of SNP’s) and Patient DNA allowed to probe stops just below the target polymorphism hybridize Patient DNA is fragmented and hybridized to chip rs100000010 rs1108985 rs1109052 Probe DNA strand is elongated one base pair that has a fluorescent tag Probe strand is Fluorescence intensity is captured, and genotype assigned elongated and substrate bp has Loss of heterozygosity = deletion or uniparental disomy of a whole chromosome unique fluorescence rs100000010 rs1108985 rs1109052 Deletion Log Ratio Patient DNA is washed away Hetero a us homp 24 % B allele Frequency AA rs100000010 rs1108985 rs1109052 Allele Frequency AB 6 - BB Fluorescence intensity captured, interpreted and Loss of Heterozygosity Hump rs100000010 rs1108985 rs1109052 reported 24 DNA Sequencing Sanger sequencing using P-32 labeled nucleotides Dideoxy chain termination, radioactive, manual, slow, ~1000 bp at a time 4 tubes required to obtain one sequence information Automated sequencing using fluorescent dideoxy nucleotides Each base a different color for automation, ~1500 bp at a time Each sequence reaction is one tube for one sequencing reaction Next Generation Sequencing (NGS) Fluorescence, lasers, computer analysis Massively parallel obtainment of sequence data fragments Need reference map, the human genome, to align sequence data fragments Next, next, next generation New technologies continue to be discovered 25 SOM.MKIII.BPM1.1.FTM.3.GNET.0077&8  Relative cost and effort to analyze Next Generation Sequencing (NGS) data - Whole Genome Sequencing (WGS) More data, more analysis, more All 6 billion base pairs (we are diploid) variants identified Terabytes of information is processed, many variants of unknown and more $$$$ significance found (both coding and non-coding genetic information) Whole Exome Sequencing (WES) 1-2% of genome that encodes for proteins Exons plus intron/exon boundary information (acceptor and donor slice site information) Targeted Gene Panels A group of genes involved in a common pathway or disease state (pathology) E.g. gene panel for cardiomyopathy Single Gene Analysis Less data, less analysis, less variants In depth analysis of a single gene found & less $ Can identify heterozygosity 26 SOM.MKIII.BPM1.1.FTM.3.GNET.0078 NGS: Massively parallel sequencing on a chip TMT 1. DNA is isolated from patient cells 2. Break DNA into fragments of similar sizes 3. Add adapters to the DNA fragments 4. Attach DNA fragments to the surface of flow channels 5. Attach DNA primer with 3’ OH group OH OH OH 27 SOM.MKIII.BPM1.1.FTM.3.GNET.0077 NGS: Fluorescent Bases Added One at a Time 1. Fluorescently labelled base pairs are substrate for synthesis reactions One base pair is incorporated into each growing chain 2. Color imaging is taken & fluorescent tag is cleaved 3. Process is repeated & computer collects data for each fragment 28 SOM.MKIII.BPM1.1.FTM.3.GNET.0077 NGS: Sequence Information from Fragments Aligned to Reference Sequence Alignment of sequence fragments compared to reference genome Sequence is determined Read depth is a measure of how many sequences were compared to reference 1 1 2 2 1 3 3 2 4 3 1 4 2 5 3 4 5 Sequencing 6 Sequencing Error Variant 7 Error 29 SOM.MKIII.BPM1.1.FTM.3.GNET.0078 NGS: In Depth Read Accuracy of sequencing is improved with more reads Allows for the determination of heterozygosity with better confidence Multiplex or massively parallel sequence acquisition: millions of sequences obtained simultaneously Can be 100X or more 30 SOM.MKIII.BPM1.1.FTM.3.GNET.0078 NGS: Variants of Unknown Significance (VUS) May or may not be disease causing Polymorphism if seen in people who do not have the disorder Sequencing may show previously unidentified variant in DNA (rare) A rare variant can be predicted to be disease causing Predicted loss of function Frame shift, non-sense, splice site, premature stop codon Variant may be in non-coding DNA region with unknown significance Sequencing whole genome will typically show many VUS’s Sequencing whole exomes will show less VUS’s Sequencing a panel of genes, even less VUS’s 31 SOM.MKIII.BPM1.1.FTM.3.GNET.0078 NGS: Gene Panels- Obtaining sequence data from targeted selection of genes Targeted Panels: During DNA preparation, DNA from the genes on panel are enriched through hybridization or PCR amplification Virtual Panels: Sequencing of genome performed, only relevant genes are analyzed Types of Panels (either targeted or virtual) Cardiovascular health Unexplained epilepsy Endocrine disorders Intellectual disability Cancer panels Fusion genes (~21 genes) Insertions/deletions (~35 genes) Gene amplification (~19 genes) Myeloid panel 32 SOM.MKIII.BPM1.1.FTM.3.GNET.0076 & 8 Cancer Gene Panels with cDNA or RNA Use of “transcriptomics” Analyze gene expression (mRNA) of a set panel of genes involved in cancer Can give numerical score that predicts prognosis or response to treatment etc. Relies on: The cDNA microarray (expression array) RNAseq (NGS for RNA) 33 SOM.MKIII.BPM1.1.FTM.3.GNET.0077  Recall cDNA microarray or Expression array Microarray has probes of reference DNA attached to a glass chip, Each spot has many of the same probe attached Tumor cells Reverse Hybridize to Isolate Label with transcriptase to Mix Microarray and Control cells RNA fluorescence cDNA scan Not present in cells Present in both cells In Normal cells only In Cancer cells only 34 SOM.MKIII.BPM1.1.FTM.3.GNET.0074 Recall RNA microarray or Expression array Microarray has probes of reference DNA attached to a glass chip, Each spot has many of the same probe attached Tumor cells Isolate Label with Hybridize to RNA fluorescence Mix Microarray and Control cells scan Not present in cells Present in both cells In Normal cells only In Cancer cells only 35 SOM.MKIII.BPM1.1.FTM.3.GNET.0074 Array CGH is becoming a first-tier clinical diagnostic test for suspected developmental disabilities/congenital anomalies (CNV) vs. G-banding Exome Sequencing is becoming a first-tier clinical diagnostic test for suspected neurodevelopmental disorders 38 Summary of Genomic Testing vs. Genetic Testing Query entire genome: Query a specific target G-band (you need to make an oligonucleotide/s specific as a probe or primers) SKY FISH ASO blot Array CGH RFLP SNP Chip PCR cDNA microarray Southern blot NGS (WGS, WES) FISH interphase & metaphase (locus specific) Sanger sequencing (sequencing a single gene) Query a specific target (isolate data collection or enrich DNA sample) NGS (panel or in-depth read of one gene) 39 SOM.MKIII.BPM1.1.FTM.3.GNET.0080 Summary of Genomic Tests Test Technique Diagnosing genetic disorders A high-throughput technique that sequences millions of DNA or Whole genome sequencing (WGS) determines complete DNA sequence for both cDNA fragments simultaneously. It allows for the detection of coding and non-coding regions Next Generation genetic variations compared to a reference genome. Whole exome sequencing (WES) focuses on sequencing protein coding regions sequencing RNA is now being sequenced but with other type of technology cDNA sequencing cDNA is made from mRNA and gives information regarding which includes nanopores expression Patient lymphocytes are cultured (grown in media) and arrested Analysis of chromosomes to detect chromosomal abnormalities like Triploidy, in metaphase by the addition of a spindle inhibitor like large deletions, duplications, translocations and inversions. Resolution, must be G-banding colchicine. Chromosomes are fixed to slide and dyed with larger than > 5 Mb, or must change banding pattern. Can be combined with FISH if Giemsa which produces characteristic banding patterns. (AT-rich a microdeletion is suspected regions stain deep purple) CGH – copy number variants (duplications or deletions but never useful A method that allows for the simultaneous analysis of thousands for translocations) of DNA sequences. It involves hybridizing DNA samples to a solid SNP – genotyping/haplotyping, UPD (when comparing to both parents) Can give Microarray support containing immobilized probes. Microarrays are used for information on duplications and deletions when a loss of heterozygosity is gene expression profiling, genotyping, and detecting copy identified number variations. Expression – compare gene expression between to populations of cells (different tissue, diseased vs. normal tissue in same organ) can be RNA or cDNA Metaphase – performed on cells that have been cultured and arrested in metaphase, can be performed on same samples prepared for G-banding A technique that uses fluorescent probes to detect specific DNA Interphase – performed on cells fixed to a slide with no need for culturing, Fluorescence In Situ sequences on chromosomes. FISH is commonly used to identify sometimes referred to as Rapid FISH Hybridization (FISH) chromosomal abnormalities, such as gene rearrangements or SKY FISH – thousands of probes designed to color each chromosome in a aneuploidies. particular color, best to see cancer rearrangements, not informative for duplications or deletions, can be metaphase or interphase DNA Polymerase chain reaction A technique used to amplify a specific DNA sequence. It involves Triplet expansion repeats, VNTR or STR fingerprinting, deletions by PCR sizing, (not a genomic test but in cycles of denaturation, annealing, and extension using DNA amplifying regions of DNA for further testing this lecture) polymerase. 40 Some specific points for students to use as study guide: Genetic & Genomic Tests Can you differentiate between indirect and direct methods of mutation detection? Can you outline the laboratory techniques, analyze, compare and contrast, and interpret results of some of the common tests that are used for molecular diagnosis of mutations Can you discuss how sizing of PCR products may be useful in genetic diagnosis Use of PCR of regions that have variable amounts of repeat expansions (such as VNTR and STR – use in gene mapping, forensics, and paternity testing diagnosis of triplet repeat disorders by Southern blot and by PCR sizing Are you able to compare and contrast the limitations and advantages of each of the molecular tests Explain to your friend how the following techniques are used to understand chromosomal abnormalities: karyotyping, chromosome banding (staining), FISH (fluorescent in situ hybridization, SKY (spectral karyotyping), array CGH (Comparative Genomic Hybridization), array SNP chips Compare, contrast, analyze and differentiate between techniques that are able to identify whole chromosome defects (aneuploidy) and chromosomal microdeletions Describe how chromosomal analysis can identify: Aneuploidy Mapping of chromosomal breakpoints Detection of subtle translocations Characterization of complex rearrangements Gains, losses and amplifications of DNA Can you describe the general concept of NGS (next generation sequencing) Discuss with your friend about nucleic acid-based microarrays cDNA microarrays (gene expression at the mRNA level) Array CGH SNP chips Compare and contrast the effects of and discuss the various types of mutation that occur in the genome including: substitution (missense, nonsense) insertion/deletion (in-frame, frameshift) splice-site amplification of repeat sequence, genomic instability regulatory mutation (promoter, regulatory element) Clinical examples include diseases involving single base mutation (sickle cell anemia), and repeat expansion disorders (Fragile X, Huntington disease, myotonic dystrophy). Other examples include trisomy syndromes such as Down syndrome (trisomy Chr. 21); Microdeletions such as DiGeorge syndrome (22q11.2 - Chr. 22 microdeletion); and 57 cancer (due to genome rearrangements and amplifications). 41 The End